Major characteristics of type 2 diabetes include impairments of insulin signaling and skeletal muscle glucose utilization, manifested by defects in IRS-1 associated PI3-kinase activity and the activation state of glycogen synthase (GS). The activity of GS is controlled, in part, through phosphorylation by glycogen synthase kinase-3 (GSK3), which inactivates GS. GSK3 can also phosphorylate IRS-1, reducing its function. We have shown that GSK3 activity is elevated in muscle from Type 2 diabetic subjects. The hypothesis to be tested in this project is that elevations of GSK3 contribute to insulin resistance. Specific aim 1 will be to determine the role of GSK3 in regulation of insulin signaling. We will delineate the impact of GSK3 manipulations on insulin signaling in cultured human skeletal muscle cells and assess correlations between GSK3 expression and insulin-stimulated IRS-1 associated PI3-K in skeletal muscle biopsies. Specific aim 2 will determine what factors of the in vivo diabetic environment lead to acquired defects of GSK3 in diabetic muscle. Human skeletal muscle cells from non-diabetic and diabetic subjects will be grown in culture and the metabolic conditions (glucose, insulin, free fatty acids, TNFalpha) selectively manipulated to recreate the diabetic metabolic phenotype and assess the impact on GSK3 expression. Specific aim 3 will obtain more specific information about the direct involvement of GSK3 in muscle cell metabolism and insulin action by use of overexpression, targeted reduction with antisense oligonucleotides, and specific inhibitors of GSK3 in muscle cells, to determine whether alterations in GSK3 can influence muscle metabolism. Specific aim 4 will fully characterize the relationship between GSK3 expression and activity in skeletal muscle and adipose tissue biopsies and in vivo insulin action and glucose disposal of lean non-diabetic, obese non-diabetic and obese Type 2 diabetic subjects. GSK3 expression and activity will be compared between muscle and fat from the same subjects. Besides basic mechanistic information about insulin signaling, these studies will reveal whether GSK3 plays a key role in the pathogenesis of Type 2 diabetes, increasing our understanding of the causes and possible treatments of insulin resistance.